Using existing, conventional, confocal microscopy, a three-dimensional specimen is scanned point by point in two-dimensional optical planes. This method is very time-consuming for capturing a three-dimensional specimen, in particular if the signal-to-noise ratio is to be optimised and reaches its limits in the case of a quick time sequence of three-dimensional images of a specimen.
WO 2004/053558 A1 discloses the method of so-called light sheet microscopy, in which an optimal image of a two-dimensional plane of the three-dimensional specimen is achieved by means of a homogenous illumination of an optical plane perpendicular to the detection lens. If a specimen is now moved through the light sheet, this results in a series of two-dimensional images and thus a three-dimensional image of the specimen. The advantages of this technique are a high imaging speed, high lateral resolution, great depth of penetration into the specimen and low excitation energy, particularly in the case of fluorescence images.
One problem with existing constructions is in particular the positioning of specimens in the light sheet. For this purpose it is simplest to secure the specimen and position it in front of the lens. Alternatively, the specimen is embedded into a cylinder, preferably in agarose with a low melting point, or is fixed in a clear container, preferably made from agarose with a low melting point or from a transparent polymer. Alternatively, cells fixed on a cover slip are held in the light sheet, see E. G. Reynaud et al., Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage, HFSP J. 2008, 2, 266-275.
This type of positioning is disadvantageous especially for large specimens, for example embryos, because they are each embedded in a matrix, which usually has different optical properties from the specimen itself and from the respective environment, for example air, water or saline solution. Furthermore, total embedding prevents the growth of biological specimens, for example embryos during time lapse images over several hours, which is particularly disadvantageous for in vivo microscopy of microscopically large specimens.
If cells are fixed on a microscope slide then they are positioned in the light sheet together with the slide. The slide is either positioned at an acute angle, approximately 45°, or parallel to the light sheet. If there is simultaneous illumination from two sides in the case of positioning at an acute angle, then an additional penetration of the slide from one of the two illumination sides results in changed optical properties. This is problematic inasmuch as, in optimal double-sided illumination, the same properties are desirable from both illumination sides.
If the slide holding the cells is orientated parallel to the light sheet, more scattered light occurs during illumination and detection near the glass surface, which brings about a poor signal-to-noise ratio. Therefore, in particular elements of a cytoskeleton, which are near the glass surface, are optically inaccessible.
A further disadvantage of existing devices for light sheet microscopy is the quick and correct positioning of the loose or embedded specimen in the light sheet. In existing constructions, a specimen is usually correctly aligned at the beginning of the measurement and (time-lapse) imaging is started. A rapid changeover between two or more specimens, i.e. so-called screening of a plurality of specimens at the same time on the same device, and their correct alignment relative to the light sheet is complex or impossible for various reasons. Firstly, each specimen has to be accurately positioned to the micron in all three spatial coordinates and this accuracy must be maintained for a long series of measurements, i.e. over several hours. Moreover, it must be ensured that all axes of rotation are maintained. The problems arising from the specifications referred to are obstructive in practice in light sheet microscopy, although they can be solved with a great deal of complexity.
Further specifications must, however, be considered. Confined spatial conditions result from the arrangement of the lens and the construction of the sample chamber, which lead to removal of the specimen from the fluid and storage outside of the sample chamber becoming necessary during the changeover of a specimen, which entails unwanted influences on a sensitive specimen, which are not acceptable especially over longer periods of time. A repetitive positioning of a series of specimens, which is accurate to the micron, for time-lapse images together with integrity of the samples cannot be ensured. Automation for non-repetitive serial imaging of a plurality of specimens or for repetitive serial imaging of a specific number of specimens over a period of time is therefore not possible.
A changeover of a detection lens and/or an illuminating lens is not easy to accomplish in the current prior art and requires, in particular for an automated change, special devices for light sheet microscopy. A suitable device for this is disclosed in DE 10 2007 018 862 A1. The disadvantage with this is that a change of lens is only possible in one axis. If more than one lens is to be used, these must be arranged in series, which normally causes problems with space. A simple change between air and immersion lenses is ruled out. If all of the lenses are immersion lenses it is necessary to arrange said lenses fully inside the specimen chamber, which is not practical in particular in the case of a plurality of lenses. Finally, revolving nosepieces, which are standard in light microscopy, cannot presently be used for light sheet microscopy.
A combination and thus a synergy of a light sheet microscope with an existing light microscope are difficult to achieve in contrast to confocal and multiphoton microscopy. Descriptions of an improved nosepiece and a combination with an upright microscope can be found in DE 10 2007 015 061 A1 and in Zanacchi et al., Live-cell 3D super-resolution imaging in thick biological samples, Nature Methods 2011, 8, 1047-1050.
In Inclined Selective Plane Illumination Microscopy Adaptor for Conventional Microscopes, Microscopy Research and Technique, published electronically on 27 Jun. 2012 under doi:10.1002/jemt.22089, F. Cutrale and E. Gratton describe a device for light sheet microscopy. Here, the same lens is used in a manner known as Highly inclined and laminated optical sheet microscopy (HILO), both as an illuminating lens and as a detection lens. The major disadvantage with this, namely the narrow field of view, is remedied here by a device for so-called Oblique plane microscopy (OPM), in which the focal plane of the image is refocused and recaptured using two additional air lenses, which are arranged at an angle relative to one another, which is not equal to 90°.